Discovery of leucine analogues of tyrocidines and tryptocidines from Brevibacillus brevis B011 through genome mining and in vitro biosynthesis

Abstract

Brevibacillus brevis B011 is an endophytic isolate from tobacco roots with significant antifungal activity, yet the metabolites responsible for this phenotype and their biosynthetic mechanisms have not been clearly resolved. Genome mining predicted 13 biosynthetic gene clusters, including nonribosomal peptide clusters for edeine, tyrocidine, and gramicidin. Gene knockout, confrontation assays and High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS) indicated that edeines is the primary determinant of antifungal activity, with tyrocidine contributing secondarily, whereas gramicidin plays a minimal role under the tested conditions. Absolute quantitative shows that the total yield of edeine is 19.2 mg/L. Based on LC–MS total ion chromatogram peak areas, the relative abundances of tyrocidines F, G, H, and I were normalized to the sum of these four derivatives as 100%, giving proportions of 25.6%, 18.4%, 34.4%, and 21.6%, respectively, with tyrocidine H being the most abundant. MS/MS putative the primary structure of edeines and four tyrocidine derivatives (each containing leucine as the third residue). The derivatives and their biosynthetic pathways were further verified through in vitro one-pot system. Comparative analysis revealed that tyrocidine F is a previously unreported derivative, whereas the remaining derivatives are consistent with reported streptocidins. Multiple sequence alignment of the adenylation (A) domains showed that TycB_M3 had only 34.7% identity with the canonical Phe-specific A domain (1AMU), showing higher similarity (39.1–56.9%) to Leu-specific A domains and exclusively recognizing leucine. Molecular docking predicted Leu-specificity-conferring residues (S401, Q404, G424, K427, S429, S591, and D593), with G424 (G301 in 1AMU) likely playing a key role in the substrate shifts from Phe to Leu. This study discovery new antimicrobial peptides through genome mining and established an antifungal phenotype-metabolite-BGC framework for B. brevis B011, providing a stronger experimental basis for its future application in plant disease biocontrol.

Graphical abstract

Highlights

• •Genome mining predicted 13 putative biosynthetic gene clusters in B. brevis B011.
• •Edeine is the major antifungal peptide, and tyrocidine is a secondary contributor.
• •Tyrocidine analogues were preliminarily structurally characterized by MS/MS.
• •Tyrocidine-related peptides were reconstituted via in vitro one-pot biosynthesis.
• •Sequence alignment and molecular docking were used to explore substrate specificity of the TycB_M3 A domain.

1. Introduction

Natural products remain a major source of bioactive scaffolds for medicine and agriculture [[1], [2], [3]]. Secondary metabolism in bacteria has certain advantages over that in plants and fungi due to factors such as a high coding density for biosynthetic genes in clusters within bacterial genomes, the absence of introns, and the ease of cultivation [4]. The genus Brevibacillus (formerly Bacillus brevis) was established in 1996 [5]. Brevibacillus spp. are commonly regarded as safe microorganisms and have been explored as probiotics, microbial cell factories, and functional microbes in biomedical and environmental remediation [[6], [7], [8]]. Notably, their potential for biocontrol of plant pathogens has gained increasing attention in recent years [[9], [10], [11], [12], [13]].

The application potential of Brevibacillus largely stems from its capacity to produce diverse secondary metabolites, especially NRPS-assembled antimicrobial peptides (AMPs) with broad-spectrum activity [14,15]. A classic example from Bacillus brevis (now reclassified within Brevibacillus and Aneurinibacillus) was the tyrothricin complex first reported by Dubos [16,17], which comprises neutral linear gramicidins (A, B, and C; collectively known as gramicidin D) and cationic cyclic decapeptides such as tyrocidines, tryptocidines, and phenycidines [[18], [19], [20], [21], [22]]. The related cyclic decapeptide gramicidin S was produced by Aneurinibacillus aneurinilyticus [23]. Early studies defined major tyrocidines (A–D) [24,25], later MS-based analyses revealed substantially broader cyclodecapeptide diversity within tyrothricin [26]. Tyrocidines show notable antiparasitic activity with relatively low mammalian toxicity and have been explored for agricultural control of plant and soil pathogens [[27], [28], [29], [30], [31], [32]]. Beyond classical tyrocidines, additional related cyclodecapeptide families (e.g., loloatins and streptocidins) further expand the structural diversity of tyrocidine-type NRPS products [33,34]. Edeine, which was first reported in 1959 [35], contains four non-proteinogenic amino acids, one glycine residue, and one polyamine moiety [36]. It not only exhibits potent antimicrobial activity against Gram-positive and -negative bacteria, mycoplasmas, and fungi and possess immunosuppressive properties [[37], [38], [39]], but also eliminate plasmid-borne resistance and inhibit cell division [40,41]. In addition to these three representative AMPs, Brevibacillus species produce a variety of AMPs, including BT [42], tostadin [43], surfactin [44], spergualin [45], and laterocidin [46], according to the non-ribosomal peptides (NRPs) database Norine (https://bioinfo.cristal.univ-lille.fr/norine/), further highlighting their value as a resource for biocontrol and medicinal applications.

Secondary metabolites are a major source of drugs and bioactive leads, and antibiotic resistance has intensified the need for new scaffolds. However, traditional bioassay-guided fractionation has become less efficient because it frequently re-isolates known compounds and yields relatively few genuinely new structures [47]. Genome mining addresses this by leveraging biosynthetic potential revealed by next-generation sequencing and biosynthetic gene clusters (BGCs) annotation, including cryptic clusters silent under standard culture conditions [[4], [48]]. With tools such as antiSMASH enabling comprehensive BGC detection, genome-informed prioritization has become a practical route for AMP discovery [49], exemplified by the identification of aurantinins B–D and surfactin-C18 from Bacillus subtilis strains [50,51].

Brevibacillus brevis B011 was an endophytic bacterium isolated in our laboratory from tobacco roots and exhibits conspicuous antifungal activity in confrontation assays, indicating potential for plant-disease biocontrol. However, the causal links among its inhibitory phenotype, the key NRPS-derived AMPs, and their corresponding biosynthetic gene clusters (BGCs) remain insufficiently defined. In this study, we employed a genome mining techniques, focusing on the NRPS-associated BGCs predicted from the B011 genome and addressed two questions: (i) what is the antifungal active compound produced by B011 under our fermentation conditions; and (ii) whether leucine incorporation in newly identified tyrocidine derivatives is associated with residue-level differences in specificity conferring code of NRPS adenylation (A) domains that govern substrate selection.

To answer these questions, we used genome mining to predictions and prioritize candidate BGCs in B. brevis B011, focusing experimental validation on NRPS-associated clusters for bioactive AMP identification. Core genes of BGCs were deleted to construct mutants using the Red/ET [52] homologous recombination technique. The primary active AMPs and their BGCs were revealed using confrontation assays, fermentation product analysis, and high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) profiling. To further validate the biosynthetic pathway and structural diversity of novel AMPs, tyrocidine was reconstructed using a one-pot in vitro enzymatic synthesis system. The established one-pot system also provides a feasible foundation for future combinatorial biosynthesis to alter specific amino acid residues in AMPs and enhance their activity. Multiple sequence alignment combined with molecular docking predicted specificity-conferring residues in the non-ribosomal peptide synthetase (NRPS) adenylation (A) domains that are responsible for substrate recognition. This study represents the first research establishing an antifungal phenotype–metabolite–BGC framework by genome mining for B. brevis B011, providing a stronger experimental basis for future application-oriented studies and its potential use in plant disease biocontrol.

2. Materials and methods

2.1. Strains, plasmids, and chemicals

Table S1 lists all of the strains and plasmids used in this study. Brevibacillus brevis B011 (NCBI accession number NZ_CP041767.1), pathogenic fungi [namely, Phytophthora parasitica var. nicotianae (tobacco black shank), Alternaria alternata (tobacco brown spot), Phytophthora capsici (Phytophthora blight of pepper), Colletotrichum scovillei (pepper anthracnose), Fusarium graminearum (Fusarium head blight of wheat), and Magnaporthe oryzae (rice blast)] and plasmid pE194 were stored in our laboratory. All primer sequences (Table S2) were obtained from Sangon Biotech Co., Ltd. (Shanghai, China), and PrimeSTAR Max DNA polymerase and markers were purchased from Takara Bio Inc. (Dalian, China). Escherichia coli strain was cultured at 37 °C in either liquid or solid Luria–Bertani (LB) medium [53], prepared using 10 g of tryptone, 5 g of yeast extract, and 1 g of NaCl per liter of distilled water, adjusted to pH 7.5. B011; and its mutants were grown under the same conditions. Fermentation cultures were grown in tryptone soy broth (TGS) medium [54,55], prepared using 15 g of tryptone, 30 g of glucose, and 5 g of NaCl per liter of distilled water, adjusted to pH 7.5. Pathogenic fungal strains were cultured on potato dextrose agar (PDA) medium [56], prepared with 200 g of peeled potatoes, 20 g of glucose, and 15 g of agar in 1 L of distilled water; this mixture was boiled, filtered, and adjusted to pH 7.5 before autoclaving.

2.2. Secondary metabolite gene cluster prediction and analysis tools

The gene cluster architecture was predicted using the AntiSMASH program (https://antismash.secondarymetabolites.org). To identify the secondary metabolite BGCs in B. brevis strain B011, NCBI accession number NZ_CP041767.1 was entered in AntiSMASH, and all extra features were selected.

2.3. Construction of B011 mutant strains

Fig. S1 illustrates the theoretical construction process for B011 mutant strains. For example, the core genes of the tyrocidine BGC were targeted for deletion.

The first step was constructing a knockout plasmid targeting the core genes of the tyrocidine BGC. Knockout vectors were engineered to contain apramycin resistance cassettes flanked by loxP sites, using Red/ET [52] homologous recombination and the Cre/loxP site-specific recombination system [57,58]. Briefly, genomic DNA from the 011-wild-type (WT) strain served as a template for amplifying two ∼1 kb homologous arms flanking the target core genes; the upstream arm (HAF) and downstream arm (HAR) were amplified using primer pairs tyrKO-HAF-F/tyrKO-HAF-R and tyrKO-HAR-F/tyrKO-HAR-R, respectively. The apramycin resistance cassette (loxM-aprA) flanked by loxP sites was amplified from the pSET152 plasmid using primers Apra1/Apra2. The temperature-sensitive vector backbone was amplified from plasmid pE194 with primers pE194-F/pE194-R. All four fragments—HAF, HAR, loxM-aprA, and pE194 backbone—were mixed (final concentration of 20 ng/μL each) and co-transformed into E. coli GB-05dir carrying the Red/ET recombination system [59,60]. Recombinant clones were selected on LB agar plates containing 20 μg/mL apramycin. Resulting plasmid pE194-tyrocidine-ko-loxM-aprA was verified by restriction enzyme digestion and DNA sequencing.

In the second step, knockout plasmid pE194-tyrocidine-ko-loxM-aprA was introduced into the 011-WT strain, and mutant strains were generated following previously described protocols [52]. Core genes in tyrocidine were successfully replaced via homologous recombination. Transformants were selected in LB liquid medium containing 10 μg/mL apramycin. Polymerase chain reaction (PCR) screening was performed using specific primers (P2/011-tyrKO-TF and P3/011-tyrKO-TR) targeting the integration site, and insertion was confirmed using DNA sequencing. The resulting mutant was designated as 011Δtyr:loxM-aprA.

In the third step, the Cre recombinase expression plasmid pDG148-Erm-Cre was introduced into 011Δtyr:loxM-aprA to excise the apramycin resistance cassette following a previously established protocol [52]. Recombinants were selected on LB medium supplemented with 2 μg/mL erythromycin. Successful excision was verified using PCR with primers 011-tyrKO-TF and 011-tyrKO-TR and DNA sequencing. Sequence analysis confirmed the presence of the expected 34-bp lox72 site, which encoded a short peptide and retained flanking sequences from the original knockout site. Thus, the apramycin resistance cassette was successfully removed, and the final mutant strain was named 011Δtyc.

Similarly, mutant strains 011Δede and 011Δgrc were obtained using the same strategy. Plasmid pE194-edeine-ko-loxM-aprA was introduced into 011Δtyc, and the double mutant 011Δede-tyc was successfully constructed following the same procedure. 011Δede-grc was also obtained using this approach.

2.4. HPLC-MS/MS analysis of tyrocidine derivatives in fermentation products

Fermentation of 011-WT and its mutant strains was performed in TGS medium as previously described [12]. Briefly, each strain was first grown overnight at 37 °C in 5.0 mL of liquid LB medium, used to inoculate 100 mL of fresh TGS medium at an initial OD₆₀₀ of 0.1, and incubated for an additional 72 h. It was then centrifuged at 9000 rpm for 10 min, and the precipitated fermentation cells were collected. The cells were mixed with 10 mL of methanol for metabolite extraction, and the mixture was incubated at 4 °C overnight and centrifuged to collect the supernatant. The extract supernatant was filtered through a 0.22-μm membrane filter, and 100 μL of the filtrate was used for HPLC-MS/MS analysis. All experiments were performed in triplicate, with parallel measurements for each replicate.

HPLC-MS/MS (Agilent 1290-6545) detection was performed following a previously described method with slight modifications [61]. Chromatographic separation was performed using an XSelect HSS T3 column (4.6 × 150 mm, 3.5 μm, 100 Å; Waters, Shanghai, China). The mobile phases were solvents A (H₂O + 0.1% TFA) and B (acetonitrile + 0.1% TFA). Gradient elution was applied as follows: 0–5 min = 55% B, 5–10 min ramp to 85% B, 10–15 min ramp to 99% B, 15–18min = 99% B, 18–20 min ramp to 5% B, and 20–23 min re-equilibration = 5% B. The column temperature was 30 °C, and the flow rate was 100 μL/min. The injection volume was 2 μL. Mass spectrometry was performed in positive ESI⁺ mode. An initial full-scan analysis (m/z 50–1800, Scan Rate 2 spectra/sec)) was conducted to survey peptide ions. Instrument Parameters: Gas Temp (320 °C), Gas Flow (8 L/min), Nebulizer (35 psig), SheathGasTemp (350), SheathGasFlow (11), Collision energy (40V). Based on the observed precursor ions and previously reported tyrocidine masses, multiple reaction monitoring transitions were subsequently established for targeted detection. The peak signal intensity corresponding to tyrocidine and its derivatives was compared across samples. The detailed detection conditions for edeine were previously described [12]. The yield of edeine A and B were determined from the peak area) by reference to the standard curve [52]. Relative abundances of tyrocidines F, G, H, and I were estimated based on their integrated peak areas from LC-MS total ion chromatograms (TIC). In the absence of authentic standards, only the relative proportions among these four derivatives were compared. The sum of their peak areas was set as 100%, and the relative abundance of each component was calculated accordingly. All analyses were performed in triplicate.

2.5. Determination of antifungal activity

The antibacterial ability of 011-WT and its mutant strains against pathogenic fungi was determined following a previous study [12]. Briefly, uniform-sized agar plugs containing actively growing fungal hyphae were placed at the center of fresh PDA plates and incubated at 28 °C. When the fungal colony had extended to a diameter of approximately 1.0 cm, 1 μL of a culture suspension of 011-WT and its mutant strains [optical density at 600 nm (OD₆₀₀) = 1.0] was spotted 1.5 cm away from the fungal margin. The plates were returned to the incubator at 28 °C until fungal growth reached the edge of the plate. The radius of the inhibition zone was measured when the fungal colony had expanded to cover the entire plate. The inhibition rate was determined following a previously described method with slight modifications [62]. The inhibition rate was calculated as follows: inhibition rate (%) = (colony radius of pathogenic bacteria in the control group − colony radius of pathogenic bacteria in the treatment group)/colony radius of pathogenic bacteria in the control group × 100%. All assays were conducted with three independent experiments, each with three technical replicates. Statistical significance among multiple groups was evaluated by one-way ANOVA followed by Tukey's multiple-comparison test. Differences were considered significant at P < 0.05.

2.6. Enzymes expression and purification

The construction of expression plasmids followed previously reported methods with modifications [61]. The tyrA gene fragment was amplified using the primer pair 99a-011-TyrA-F/99a-011-TyrA-R, which included 45-bp overlaps with the pTrc99a vector backbone (amplified using primers 99a-F/99a-R) to produce C-terminal His₆-tagged TyrA. The resulting fragments were assembled using Red/ET homologous recombination [52], yielding plasmid pTrc99a_TyrA. The tyrB gene was amplified in three overlapping fragments using primer pairs 99a-011-TyrB-F/TyrB-1, TyrB-2/TyrB-3, and TyrB-4/99a-011-TyrB-R and assembled with the pTrc99a backbone using the same recombination strategy to construct pTrc99a_TyrB encoding His₆-tagged TycB. Similarly, pTrc99a_TyrC was generated to express His₆-tagged TycC. The tyrC gene was amplified in five overlapping fragments using primer pairs 99a-011-TyrC-F/TyrC-1, TyrC-2/TyrC-3, TyrC-4/TyrC-5, TyrC-6/Tyr7, and TyrC-8/99a-011-TyrC-R and assembled with the pTrc99a backbone using Red/ET recombination. The assembled DNA mixtures were electroporated into E. coli GB-05dir harboring the Red/ET recombination system using a Pulse Cell Transfection System [59,60,63,64]. Transformants were selected on LB agar plates supplemented with 100 μg/mL ampicillin. All recombinant plasmids were verified via restriction enzyme digestion and sequencing.

Protein expression and purification were performed based on a previously reported protocol with slight modifications [61]. Escherichia coli BL21 (DE3) cells harboring the expression plasmids were revived from glycerol stocks stored at −80 °C and cultured overnight at 37 °C in 10 mL of LB medium supplemented with 100 μg/mL ampicillin. The overnight culture (1% v/v) was used to inoculate 800 mL of fresh LB medium (in a 2-L baffled flask) supplemented with 100 μg/mL ampicillin and incubated at 37 °C with shaking at 180 rpm until the OD₆₀₀ reached 2.0. Protein expression was induced by adding 0.2 mM isopropyl β-d-1-thiogalactopyranoside (IPTG, final concentration) and then incubating at 20 °C for 20 h. Cells were harvested by centrifugation at 8000 rpm for 10 min at 4 °C and resuspended in lysis buffer containing 50 mM Tris-base, 100 mM NaCl, 5 mM MgCl₂, and 5% (v/v) glycerol (pH 7.5). Cell disruption was performed using an ultrasonic processor (160 W, 30 min, 5 s on/5 s off), and the lysate was clarified by centrifugation at 9000 rpm for 20 min at 4 °C. The resulting supernatant was loaded onto a Ni-NTA column and washed with buffer containing 50 mM NaH₂PO₄, 100 mM NaCl, and 20 mM imidazole (pH 8.0). The target protein was eluted with elution buffer containing 50 mM NaH₂PO₄, 100 mM NaCl, and 250 mM imidazole (pH 8.0). The eluted fractions were concentrated and desalted using ultrafiltration tubes and buffer-exchanged into storage buffer containing 50 mM HEPES, 100 mM NaCl, 5 mM MgCl₂, and 5% (v/v) glycerol (pH 7.5). Purified proteins were aliquoted, flash-frozen in liquid nitrogen, and stored at −80 °C. The protein concentrations were determined using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and corrected using the theoretical extinction coefficient calculated in ProtParam (https://web.expasy.org/protparam/). Protein purity was assessed via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

2.7. In vitro tyrocidine biosynthesis

A one-pot enzymatic reaction was performed based on a previously described protocol with slight modifications [61]. Reactions were carried out in Bis-Tris propane buffer (100 mM, pH 7.5) supplemented with 5 mM MgCl₂, 100 mM NaCl, 1 mg/mL BSA (Sigma-Aldrich, St. Louis, MO, USA), 4 mM TCEP, 0.1 U/μL inorganic pyrophosphatase (from baker's yeast, Merck, Rahway, NJ, USA), and 50 mM ATP. The enzymatic components included TycA (10 μM), TycB (10 μM), TycC (10 μM), CoA (300 μM), and Sfp (6 μM). Substrate amino acids were added at the following final concentrations: 4 mM L-Phe, 2 mM L-Pro, 2 mM L-Asn, 2 mM L-Gln, 2 mM L-Tyr, 2 mM L-Val, 2 mM L-Orn, 4 mM L-Leu, and 2 mM L-Trp. The reaction was initiated by the addition of ATP and incubated overnight at 37 °C in a water bath. Upon completion, each 200 μL reaction mixture was quenched by adding 1.6 mL methanol, followed by vortexing and centrifugation at 12,000 r/min for 10 min to remove precipitated proteins. The supernatants were then vacuum frozen and concentrated to a final volume of 200 μL and analyzed by HPLC-MS/MS.

2.8. Structural modeling, molecular docking, and multiple alignment

The three-dimensional (3D) structures of A domain (the amino acid sequence is shown in Table S3) in 011_TycB_M3 (WP_237900949.1) and its mutant were predicted using AlphaFold 3 (https://golgi.sandbox.google.com), and the model quality was assessed using the SAVES v6.0 server (https://saves.mbi.ucla.edu/). The molecular structures of leucine (Leu), phenylalanine (Phe), and adenosine monophosphate (A_MP) were obtained from the PubChem database (http://pubchem.ncbi.nlm.nih.gov). Molecular docking was performed using AutoDock Vina [65] to investigate the substrate binding affinity. The binding site was defined as a cubic grid box with dimensions of 27.75 × 26.25 × 25.5 (length × width × height), and the center was set at coordinates (−5.849, 4.508, 4.749) based on prior structural information from the 1AMU active pocket and multiple sequence alignment. Active pocket and ligand docking simulations were performed for Leu/Phe and A_MP with both WT and mutant 011_TycB_M3 structures. The docking conformation (docking complex) with the lowest binding energy was selected for further analysis using PyMOL. The resulting complexes were analyzed to evaluate the binding interactions and structural compatibility. Visualization and interaction analysis were rendered in 2D/3D formats to facilitate the interpretation of substrate recognition mechanisms. Multiple sequence alignment was performed using MEGA X and visualized using ESPript 3.0 (http://espript.ibcp.fr/ESPript/ESPript/) to identify conserved residues relevant to substrate specificity.

3. Results

3.1. Genome analysis of B. brevis B011

To identify the secondary metabolite BGCs in B. brevis B011, we entered NCBI accession number NZ_CP041767.1 in the bioinformatic tool AntiSMASH, which enabled the rapid genome-wide identification, annotation, and analysis of secondary metabolite biosynthesis gene clusters in B. brevis B011. There were 13 secondary metabolite BGCs in the B. brevis B011 genome (Fig. 1). Among them, four clusters showed high homology (≥80%) with known functional BGCs: Cluster 2 petrobactin, Cluster 5 tyrocidine, Cluster 6 gramicidin, and Cluster 7 edeine. Cluster 11 shared only 25% sequence similarity with known functional clusters, and eight BGCs remained unannotated (Clusters 1, 3, 4, 8, 9, 10, 12, and 13).

Fig. 1.

Schematic representation of the thirteen secondary metabolite biosynthetic gene clusters identified in Brevibacillus brevis B011. The potential BGCs were predicted using the antiSMASH web server. Each number in the overview figure corresponds to a distinct biosynthetic gene cluster. The prediction indicates the product type, cluster size, the most similar known gene cluster, and the degree of similarity. Genes with different predicted functions are indicated by color-coded blocks, as defined in the legend.

Cluster 2 was predicted to be responsible for siderophore biosynthesis (petrobactin). Iron is an essential element for all living organisms and plays critical roles in electron transport, oxygen metabolism, and enzyme catalysis [66]. Microorganisms have developed various strategies to survive in environments with limited iron availability and to obtain iron, with siderophore production being considered the most effective. Under iron-deficient conditions, microbes secrete siderophores to bind ferric ions (Fe³⁺) in their surroundings with high affinity. Subsequently, the iron–siderophore complexes are taken up into the cell through specialized uptake systems, enabling microbes to meet their iron needs [67]. It has been experimentally demonstrated that iron bioavailability from a number of discrete iron sources increases due to the production of the siderophore petrobactin [68].

Tyrothricin was a peptide antibiotic complex comprising linear gramicidins and cyclic decapeptide tyrocidines, and represents one of the earliest antibiotics applied clinically for topical use [69]. Both tyrocidines and gramicidins are nonribosomal peptides (NRPs) synthesized by large, multi-modular nonribosomal peptide synthetases (NRPSs) [14]. Their biosynthesis is encoded by dedicated NRPS gene clusters. The organization of these clusters determines peptide structure and diversity [70,71]. Below, we describe the NRPS gene cluster associated with tyrocidine/gramicidin/edeine biosynthesis.

Cluster 5 was predicted to be responsible for tyrocidine biosynthesis because of the high similarity to known tyrocidine synthetic gene clusters in B. brevis (Fig. S1). The BGC structures were intact, and phenomena such as missing or rearranged gene elements were not found. Tyrocidines, cyclic decapeptide antimicrobial peptides, exhibit a multifaceted, multi-target mechanism of action [72]. Instead of functioning exclusively as agents that form pores in membranes, tyrocidines are suggested to create amphipathic dimers or more complex structures that engage with bacterial membranes in a non-classical manner [73]. Beyond membrane disruption, tyrocidines have the ability to penetrate the cytoplasm and engage with targets inside the cell, collectively enhancing their bactericidal efficacy [74].

Cluster 6 was predicted to be responsible for gramicidin biosynthesis. Gramicidins comprise two structurally and biosynthetically distinct groups: the linear pentadecapeptide gramicidins and the cyclic decapeptide gramicidin S. Linear gramicidins are highly hydrophobic pentadecapeptides with N- and C-terminal blocking groups and an alternating D- and l-amino acid sequence, enabling the formation of a right-handed β-helix–like transmembrane structure. In lipid membranes, two gramicidin monomers associate head-to-tail to form a cation-conducting channel spanning the bilayer, with tryptophan residues positioned at the membrane–water interface to stabilize ion transport [19,75,76]. Gramicidin S is a cyclodecapeptide synthesized by an NRPS system with an assembly logic and module organization distinct from those of the NRPS pathways producing linear gramicidins. Notably, the NRPS assembly line responsible for gramicidin S biosynthesis shares five modules with the tyrocidine synthetase, exhibiting identical substrate specificity for the Val-Orn-Leu-D-Phe-Pro pentapeptide moiety that is duplicated in both cyclodecapeptides [25,77]. It promotes wound healing and rarely induces resistant strains when combined with gramicidin to form the topical antibiotic tyrothricin, making it a promising next-generation antibiotic scaffold [69].

Cluster 7 was predicted to be responsible for edeine [NRPS and polyketide synthase (PKS) hybrid] biosynthesis because of the high similarity to the known synthetic edeine gene clusters from B. brevis X23 [52] and Vm4 [36]. It is composed of multiple non-proteinogenic amino acid residues, including β-tyrosine or β-phenylalanine, isoserine, 2,3-diaminopropanoic acid (DAPA), 2,6-diamino-7-hydroxyazelaic acid (DAHAA), one glycine, and a unique (guanyl)spermidine moiety. Edeines are produced as a mixture of four main congeners—edeines A, B, D, and F—each occurring in α and β isomeric forms. These isomers differ in the specific site of peptide bond formation between the carboxyl group of β-isoserine and the amino groups of DAPA, resulting in structural variation between the α- and β-forms [36].

Three other NRPS-type clusters (clusters 8 and 13) were found to be dissimilar to any known secondary metabolites’ synthetic gene clusters in the AntiSMASH database. Cluster 11 was predicted as aurantinins but with low similarity. Aurantinins are secondary metabolites with antibacterial activities that have a high potential for novel biosynthesis [78].

Several BGCs were predicted in the genome, some of which exhibited features characteristic of ribosomally synthesized and post-translationally modified peptides (RiPPs) [79] and terpenes [80] in natural products, although none showed significant similarity to known secondary metabolite BGCs in the AntiSMASH database. Among them, Cluster 1 was annotated as an RiPP recognition element (RRE)-containing cluster. RREs are conserved structural motifs commonly found in prokaryotic RiPP BGCs, and they are responsible for delivering the precursor peptide to the peptidase and activating the enzyme in a leader peptide-dependent manner [81]. Clusters 4 and 12 were classified as RiPP-type clusters. Clusters 3 and 10 were annotated as terpene-type clusters, and Cluster 9 was annotated as a type III PKS [82]. Despite these annotations, none of the identified clusters showed similarity to known secondary metabolite BGCs in the AntiSMASH database, suggesting the presence of novel or highly divergent biosynthetic pathways. However, these clusters are not the focus of this study and will be investigated in future work.

Taken together, these findings suggest that biocontrol agent B. brevis B011 has the potential to synthesize AMPs, such as tyrocidine, gramicidin, and edeine.

3.2. Construction of the mutant strains

To identify the BGCs corresponding to each AMP, we constructed a series of single and double mutant strains lacking the ability to produce one or more specific AMPs. The construction strategy is illustrated in Fig. S2. The knockout vector pE194-tyrocidine-ko-loxM-aprA was introduced into strain 011-WT via electroporation to disrupt the core genes in the tyrocidine biosynthetic pathway, enabling homologous recombination. Transformants were screened using PCR with two primer pairs (011-tyrKO-TF/P2 and P3/011-tyrKO-TR), yielding bands of the expected size, and the sequencing results confirmed no mutations (Fig. S3A). The verified mutant strain was named 011Δtyc:loxM-aprA.

A plasmid encoding Cre recombinase was introduced into antibiotic-resistant strain 011Δtyc:loxM-aprA. Transformants were screened using PCR with primers 011-tyrKO-TF and 011-tyrKO-TR (Fig. S3B). The amplified fragment was approximately 3500 bp in strains containing the Apra^R resistance cassette and approximately 2500 bp following excision of the cassette, which is consistent with the ∼1000 bp size of the ApraR marker. These results were in line with our expectations. Sequencing of the PCR products confirmed the absence of unintended mutations, showing the successful removal of the ApraR marker from the 011Δtyc:loxM-aprA genome, and the resulting strain was named 011Δtyc. Using the same strategy, additional mutant strains, such as 011Δede and 011Δgra, were successfully constructed.

Edeine knockout plasmid pE194-edeine-ko-loxM-aprA was introduced into strain 011Δtyc via electroporation, disrupting the core genes responsible for edeine biosynthesis through homologous recombination. PCR screening of the transformants using two primer pairs (011-edeKO-TF/P2 and P3/011-edeKO-TR) yielded bands of the expected sizes (Fig. S3C). Sequencing confirmed the absence of unintended mutations (Fig. S3D), and the successfully constructed double mutant was designated 011Δede-tyc. Additional mutant strains, such as 011Δede-gra, were obtained using this same methodology.

3.3. Discovery of antimicrobial peptides and their BGCs with antifungal activity

Bioinformatic predictions indicated that 011-WT has the potential to synthesize various AMPs, such as tyrocidine, edeine, and gramicidin. All three AMPs are known to exhibit inhibitory effects against plant pathogenic fungi [12,[28], [29], [30], [31], [32]]. Due to differences in the types/number of AMPs produced by mutant strains, there may also be differences in their antibacterial activities against plant pathogenic fungi. We selected pathogenic fungi of important crops as antagonistic targets, including Phytophthora parasitica var. nicotianae, Alternaria alternata, Phytophthora capsici, Colletotrichum scovillei, Fusarium graminearum, and Magnaporthe oryzae, to systematically evaluate the antibacterial spectrum of these AMPs. By comparing the differences in antibacterial activity among mutant strains, the antibacterial spectrum of each AMP, along with the corresponding BGCs, was initially revealed, laying the foundation for a deeper analysis of the multiple antibacterial mechanisms of 011-WT.

Inhibition assays (Fig. 2A–F) demonstrated that 011-WT exhibited excellent inhibitory activity against all six pathogenic fungi, with inhibition rates (Fig. 2G) ranging between 15.5 and 29.1%, suggesting that it has the ability to synthesize AMPs. The gramicidin-deficient mutant 011Δgra retained an antifungal profile (15.8–29.8%) similar to that of 011-WT, with no statistically significant difference in the sum of the inhibition rate (SIR). This suggests that 011Δgra and 011-WT may not produce gramicidin under the tested conditions and that its antifungal activity is attributed to other AMPs. Tyrocidine-deficient mutant 011Δtyc showed a slight reduction in SIR (inhibition rate of 10.8–26.7%), indicating that tyrocidine plays a secondary role in the antifungal activity. In contrast, edeine-deficient strain 011Δede showed a significantly reduced SIR (inhibition rate of 5.7–22.3%), suggesting that edeine was a major contributor to the antifungal activity.

Fig. 2.

Comparative antifungal activity of 011-WT and mutants against six phytopathogenic fungi. (A–F) Dual-culture confrontation assays picture of 011-WT and mutants against pathogenic fungi. The name of the pathogenic fungus species was indicated below the picture. (G) Statistical chart of pathogenic fungal inhibition rates (%). Inhibition rates were shown as the mean of three biological replicates. Error bars indicate standard deviations (SD). Different letters indicate statistically significant differences between groups (P < 0.05), whereas bars sharing the same letter are not significantly different, and the same below.

Double mutant analysis further confirmed these AMPs. Double knockout strain 011Δede-gra (inhibition rate of 5.2–20.6%) showed a non-significant difference in SIR compared with 011Δede, emphasizing the minimal effect of gramicidin and suggesting that it may only produce tyrocidine. As expected, 011Δede-tyc simultaneously lost its inhibitory capacity against these six pathogenic fungi and exhibited a complete loss of antifungal activity, suggesting that it lost the ability to produce tyrocidine and edeine.

Therefore, under these conditions, 011-WT may produce the main AMP edeine and the secondary AMP tyrocidine while producing little to no gramicidin and localizing to the BGCs of each AMP.

3.4. Identification of edeine using HPLC-MS/MS

Considering the confrontation assay results for 011-WT and its mutant strains, edeine is likely the major antimicrobial compound produced by B. brevis B011. Fermentation broths of 011-WT and its mutant strains were subjected to HPLC-MS/MS to confirm the primary structure of the AMP edeine. The HPLC profiles were consistent with our expectations (Fig. 3A), as the edeine-specific peak was absent in 011Δede, 011Δede-gra, and 011Δede-tyc but clearly detected in 011-WT, 011Δgra, and 011Δtyc. The target peaks for the edeines appeared around 1.1 and 1.2 min. The electrospray ionization mass spectrometry (ESI-MS) spectrum showed molecular ions at m/z values of 755.4433 [M+H]⁺ and 777.4255 [M+Na]⁺ for edeine A and 797.4655 [M+H]⁺ and 819.34469 [M+Na]⁺ for edeine B (Fig. 3B and C). The error between the theoretical and actual values of the mass spectrometry data was less than 5 ppm. Based on the previously [52] established standard curves relating integrated peak area to concentration, the absolute yields in B011-WT were 17.7 mg/L for edeine A and 1.5 mg/L for edeine B, giving a total of 19.2 mg/L (Table S4).

Fig. 3.

The HPLC-MS/MS analysis was performed to compare the metabolite profiles of the 011-WT and its mutant strains. (A) HPLC analysis of edeines in the fermentation broths of various strains. (B) and (C) ESI-MS showed molecular ions at m/z values 755.4433 [M+H]⁺ and 777.4255 [M+Na]⁺ for edeines A, at m/z values 797.4655 [M+H]⁺ and 819.34469 [M+Na]⁺ for edeines B. The [M+H]⁺ theoretical m/z values for edeine A and edeine B are 755.4410 and 797.4628, respectively; the corresponding [M+Na]⁺ values are 777.4230 and 819.4448. (D) MS/MS spectrum of edeine A with a precursor ion at m/z 755.4410 ([M+H]⁺). Major fragment ions are labeled and correspond to predicted cleavages within the edeine A structure. (E) MS/MS spectrum of edeine B showing the fragmentation pattern of the precursor ion at m/z 797.4628 ([M+H]⁺). Characteristic fragments confirm the presence of the guanylspermidine moiety.

The main difference in the N-terminal structure between edeines A and B was that edeine A contained a spermidine moiety, whereas edeine B contained a guanylspermidine group [36]. MS/MS fragmentation analysis was performed on the corresponding HPLC peak to confirm the identity of edeine and elucidate its structural features. For edeine A, the MS/MS spectrum (Fig. 3D) revealed a set of characteristic fragment ions, including the following: m/z 147.0435, corresponding to β-tyrosine (showing the loss of an NH₃ group); m/z 174.0865, representing the isoserine and 2,3-diaminopropionic acid (DAPA)-containing moiety; and a major ion at m/z 593.2583, attributed to a partial backbone fragment. Additionally, N-terminal spermidine fragments were observed at m/z 146.1644, 203.1878, 419.2971, 487.3345, and 592.3755, corresponding to the spermidine moiety at the N-terminus, consistent with the known structure of edeine A. A similar fragmentation pattern was detected for edeine B (Fig. 3E), including m/z 147.0438 (β-tyrosine, corresponding to the loss of an NH₃ group), m/z 234.0746 (β-tyrosine-isoserine fragment), and 337.1531 (β-tyrosine-isoserine-DAPA fragment). However, distinct fragments at m/z 188.1864, 245.2081, 461.3192, 547.3660, and 634.3960 were observed, corresponding to the guanylspermidine group and differentiating it from edeine A. The presence of these fragment ions confirms the guanidinated modification on the N-terminus of edeine B. Taken together, these findings suggest that B. brevis B011 produces edeines A and B, and the primary structure of edeine was putative based on MS/MS fragmentation patterns.

3.5. Identification of new tyrocidine derivatives using HPLC-MS/MS

Confrontation assays comparing 011-WT and its mutant strains showed that tyrocidine was also a major contributor to the antifungal activity of B. brevis B011. Extract supernatant from the 011-WT and mutant strains was subjected to HPLC-MS/MS analysis to confirm the identity and structural characteristics of tyrocidine. Previously reported tyrocidines include tyrocidines A, B, C, D, and E [14,25,83]. The amino acid residue sequences of tyrocidines A, B, C, D, and E were cyclo(DPhe¹-Pro²-Phe³-DPhe⁴-Asn⁵-Gln⁶-Tyr⁷-Val⁸-Orn⁹-Leu¹⁰), cyclo(DPhe¹-Pro²-Trp³-DPhe⁴-Asn⁵-Gln⁶-Tyr⁷-Val⁸-Orn⁹-Leu¹⁰), cyclo(DPhe¹-Pro²-Trp³-DTrp⁴-Asn⁵-Gln⁶-Tyr⁷-Val⁸-Orn⁹-Leu¹⁰), cyclo(DPhe¹-Pro²-Trp³-DTrp⁴-Asn⁵-Gln⁶-Trp⁷-Val⁸-Orn⁹-Leu¹⁰), and cyclo(DPhe¹-Pro²-Phe³-DPhe⁴-Asn⁵-Gln⁶-Phe⁷-Val⁸-Orn⁹-Leu¹⁰), respectively. Tyrocidine D was renamed to tryptocidine C by Barber et al. [26]. Tyrocidine E was renamed to Phenycidine A by Vosloo et al. [84]. The key differences were due to variations in the amino acid residues at positions 3, 4, and 7 (Fig. 4A). HPLC-MS analysis using extracted ion chromatograms (EIC) failed to detect any characteristic peaks corresponding to tyrocidine A, B, C, D, or E. However, base peak chromatogram (BPC) analysis revealed four consecutive target peaks (Fig. 4B) within an m/z range of 1200–1400, with retention times of 6.9, 7.3, 7.6, and 7.9 min. These findings suggest the presence of novel tyrocidine derivatives. Based on the increasing order of their m/z ratios, these compounds were tentatively named tyrocidines F, G H, and I. MS analysis (Fig. 4C) showed that tyrocidine F exhibited [M+H]⁺ and [M+Na]⁺ ions at m/z 1236.6779 and 1258.6578, respectively, tyrocidine G at m/z 1259.6944 ([M+H]⁺) and 1281.6752 ([M+Na]⁺), tyrocidine H at m/z 1275.6888 ([M+H]⁺) and 1297.6760 ([M+Na]⁺), and tyrocidine I at m/z 1298.7048 ([M+H]⁺) and 1320.6849 ([M+Na]⁺). Based on these results, we propose that B. brevis B011 does not produce tyrocidine A, B, C, D, or E but instead synthesizes a series of novel tyrocidine derivatives, named F, G, H, and I. Relative abundances of tyrocidine derivatives were estimated from LC–MS TIC peak areas. In 011-WT, the raw integrated peak areas for tyrocidines F, G, H, and I were 3.2 × 10^7, 2.3 × 10^7, 4.3 × 10^7, and 2.7 × 10^7, respectively (Table S4). These values were normalized to the sum of the four derivatives as 100% (F + G + H + I = 100%), they contributed for 25.6%, 18.4%, 34.4%, and 21.6%, respectively, with tyrocidine H being the most abundant. In subsequent work, we aim to biosynthetically produce these newly identified compounds and elucidate their primary structures.

Fig. 4.

The HPLC-MS analysis was performed to compare the metabolite profiles of the 011-WT and its mutant strains. (A) The amino acid residues sequence of cyclic decapeptides tyrocidine A, B, C, D, and E. Each compound shares the characteristic cyclic decapeptide scaffold of tyrocidines but differs in amino acid composition (highlight). The molecular formula, exact molecular weight, and theoretical [M+H]⁺ values were display. (B) HPLC analysis of tyrocidine new derivatives in the extract supernatant of various strains. (C) MS analysis of tyrocidine F, G, H, and I. The newly identified derivative compounds exhibited the following mass-to-charge ratios (m/z, [M+H]⁺ and [M+Na]⁺).

3.6. In vitro biosynthesis and primary structure elucidation of tyrocidines F, G, H, and I

Fermentation extracts were complex and the putative tyrocidines F–I were low-abundance with crowded peaks, complicating MS/MS interpretation. We therefore established a one-pot in vitro reconstitution of the Tyc NRPS system using purified TycA (128.8 kDa), TycB (408.9 kDa), and TycC (731.1 kDa) with defined substrates to generate products in a cleaner background for MS/MS confirmation (Fig. 5A). This enabled structural support for the newly identified tyrocidine derivatives (tyrocidines F–I) and strengthened their biosynthetic linkage to TycA/B/C. Cyclized tyrocidine derivatives were generated after incubation with the necessary amino acid substrates and cofactors (Fig. 5B). The biosynthesized compounds exhibited distinct peaks in the BPC (m/z range of 1200–1400), with retention times of 7.1, 7.5, 6.5, and 6.8 min and m/z values ([M+H]⁺ values) of 1236.6782, 1259.6942, 1275.6891, and 1298.7045 for tyrocidines F, G, H, and I, respectively, matching those of the corresponding metabolites detected in the extracted fermentation broth supernatant of B. brevis B011. No expected target peaks were observed when ATP, related proteins, or heat-denatured proteins were omitted from the one-pot reaction, confirming that tyrocidines F–I are authentic products of the tyrocidine biosynthetic pathway.

Fig. 5.

In vitro biosynthesis of novel tyrocidine derivatives. (A) SDS-PAGE gel showing the purified NRPSs, TycA (128.8 kDa), TycB (408.9 kDa), and TycC (731.1 kDa). Distinct bands corresponding to the expected molecular weights indicate successful expression and purification of each enzyme. (B) HPLC-MS analysis of one-pot reaction products. HPLC-MS analysis confirmed the presence of the expected target peaks corresponding to tyrocidine F–I, with observed retention times of 7.1, 7.5, 6.5, and 6.8 min, and [M+H]⁺ values of 1236.6782, 1259.6942, 1275.6891, and 1298.7045, respectively. Control reactions lacking ATP, essential NRPSs, or containing heat-denatured proteins did not yield any of the target peaks, indicating the requirement of active enzymes and complete reaction components for successful synthesis. (C) Proposed primary structures of tyrocidine F–I. Confirmed primary amino acid sequences of the newly identified tyrocidine derivatives F–I, based on MS/MS analysis. Different amino acid residues were highlighted. (D) Schematic representation of the NRPS assembly line responsible for tyrocidine biosynthesis. The pathway involves the sequential action of three large NRPSs—TycA, TycB, and TycC (blue arrow) —each composed of multiple modules that recognize, activate, and incorporate specific amino acids substrate into the growing peptide chain. NRPS domains are labeled as follow: C condensation, A adenylation, T thiolation, E epimerization, and TE thioesterase. Domain orchestrating the stepwise assembly and cyclization of the cyclic decapeptide tyrocidine.

Referencing the reported primary structures of tyrocidines A–E, we proposed the primary structures of the newly discovered derivatives F–I (Fig. 5C). The amino acid residue sequences of tyrocidines F, G, H, and I were cyclo(DPhe¹-Pro²-Leu³-DPhe⁴-Asn⁵-Gln⁶-Tyr⁷-Val⁸-Orn⁹-Leu¹⁰), cyclo(DPhe¹-Pro²-Leu³-DPhe⁴-Asn⁵-Gln⁶-Trp⁷-Val⁸-Orn⁹-Leu¹⁰), cyclo(DPhe¹-Pro²-Leu³-DTrp⁴-Asn⁵-Gln⁶-Tyr⁷-Val⁸-Orn⁹-Leu¹⁰), and cyclo(DPhe¹-Pro²-Leu³-DTrp⁴-Asn⁵-Gln⁶-Trp⁷-Val⁸-Orn⁹-Leu¹⁰), respectively. The mass deviations between the observed and calculated [M+H]⁺ values were within 5 ppm, strongly supporting the proposed structural assignments. Tyrocidines F, G, H, and I shared a common feature—the third amino acid residue was consistently substituted with Leu. The differences among these derivatives were additional substitutions at the fourth and seventh positions, where distinct amino acid residues were introduced. MS/MS analysis was performed to further verify their primary structures.

MS/MS analysis confirmed the identities of tyrocidines F, G, H, and I based on their [M+H]⁺ values and diagnostic fragmentation patterns, which exhibited mass deviations within 5 ppm of theoretical values. For tyrocidine F (Fig. S4), characteristic ions corresponding to the Leu³ residue were observed at m/z 211.1430, 375.2016, 472.2554, 732.4039, 976.5231, and 1089.6053, and neutral losses of NH₃ (m/z 1219.6558) and H₂O (m/z 1218.6626) were also revealed. Tyrocidine G (Fig. S5) produced fragment ions related to Leu³ and Trp⁷ at m/z 788.4045, 885.4602, 902.4837, 999.5376, 1032.5291, and 1145.6115 as well as neutral losses of NH₃ (m/z 1242.6716) and H₂O (m/z 1241.6786). For tyrocidine H (Fig. S6), ions at m/z 300.1694, 511.2645, 639.3232, 802.3863, 1048.5241, and 1162.6071 were consistent with Leu³ and Trp⁴, accompanied by losses of NH₃ (m/z 1258.6657) and H₂O (m/z 1257.6734). Tyrocidine I (Fig. S7) showed fragment ions indicative of Leu³, Trp⁴, and Trp⁷ at m/z 528.2565, 899.4765, 924.4710, 941.4953, 1085.5553, 1151.6311, and 1185.6231, with corresponding losses of NH₃ (m/z 1281.6821) and H₂O (m/z 1280.6886). Overall, the [M+H]⁺ values of the biosynthetic products were consistent with those of tyrocidines F, G, H, and I, and the MS/MS fragmentation patterns putative their primary structures.

These results indicate that the one-pot in vitro biosynthesis system successfully reconstituted the tyrocidine biosynthetic pathway and produced novel tyrocidine derivatives (Fig. 5D), consistent with the results obtained from the extract supernatant analyzed as described above. This approach verifies the proposed primary structures and provides a valuable tool for future mechanistic studies and biosynthetic engineering of tyrocidine-like AMPs.

3.7. Probing the substrate specificity of 011_TycB_M3 via sequence alignment and molecular docking

A common feature of the four newly identified derivatives, tyrocidines F–I, is that the third amino acid in their primary structures is Leu, indicating a potential change in substrate specificity of the A domain in 011_TycB_M3. The shift in substrate specificity of the A domain prompted us to further investigate the causes underlying this alteration.

The crystal structure 1AMU encodes an A domain that specifically recognizes and activates Phe. Residues 235, 236, 239, 278, 299, 301, 322, 330, 331, and 517 were identified as key specificity-conferring codes of the A domain substrate (Fig. 6A and S8) [85,86]. Amino acid sequence alignment (Figs. S9 and S10) revealed that the A domain of 011_TycB_M3 shared only 34.7% identity with 1AMU (GrsA). However, it exhibited high similarity to the A domain of 011_TycC_M10 (56.9%) and moderate similarity to Plu3263_M5 (42.1%), M4 (40.1%), and M2 (39.1%). These findings suggest that 011_TycB_M3 is more likely to recognize and activate Leu than Phe. This hypothesis is supported by our experimental evidence, which confirmed that 011_TycB_M3 exclusively recognizes and activates Leu as its substrate. The G301 residue might be the key specificity-conferring code enabling 011_TycB_M3 to recognize and activate Leu rather than Phe. Furthermore, residues D240, G322, and S328 in TycB_M4 (Fig. S11) as well as A236, L239, A259, G301, E322, S328, and S329 in TycC_M7 (Fig. S12) may confer substrate selectivity for Phe or Trp and Tyr or Trp, respectively.

Fig. 6.

3D model view of conserved residues underlying NRPS A domain substrate specificity. (A) Key specificity-conferring codes within the A domain of the 1AMU crystal structure that determine amino acid substrate recognition and activates. (B) 3D schematic illustrating the interaction between TycB_M3 and the Leu–A_MP complex. Hydrogen bonds were shown as yellow dashed lines. Red with bolded numbers correspond to amino acid residue positions in the multiple sequence alignment (the blue arrow in Fig. S10). Among them, G301 corresponds to G424 in 3D schematic illustrating of modeling. The upper right corner displays the surface representation of the complex.

To further explore the mechanism of substrate recognition, molecular docking was performed using AutoDock Vina among 011_TycB_M3 (Leu) (hereafter referred to as TycB_M3, the amino acid sequence for modeling; Table S3), its site-directed mutagenesis mutant (G301A, G301 corresponds to G424 in 3D schematic illustration of modeling), and selected ligand substrates (Leu or Phe together with A_MP). The 3D structures of TycB_M3 and G301A were predicted using AlphaFold3 and visualized using PyMOL (Fig. 6B and S14). The structural quality was assessed via the SAVES v6.0 server using multiple validation tools. The Verify 3D analysis showed that 68.22% of residues had a 3D-1D score ≥0.1 (Fig. S13A). However, ERRAT evaluation revealed an overall quality factor of 98.302 (considered excellent when >95), indicating a model structure with a highly reliable resolution (Fig. S13B). Ramachandran plot analysis via PROCHECK revealed that 93.5% of residues were in the most favored regions, 6.3% in additional allowed regions, and only 0.2% in disallowed regions (Fig. S13C), confirming the high quality of the 3D model for molecular docking.

The docking complexes were subsequently visualized using PyMOL. For TycB_M3 with Leu, the binding conformation exhibiting the lowest binding energy (−10.3 kcal/mol) was selected to construct the 3D structure of the active site, as illustrated in Fig. 6B. The ligand Leu formed hydrogen bonds with key residues Lys427, Ser429, Ser591, Gly592, and Asp593 within the TycB_M3 binding pocket. The docking complexes of TycB_M3 with Leu revealed the Leu substrate binding pockets and specificity-conferring codes of the A domains in 011_TycB_M3. The Leu substrate specificity-conferring codes S401, Q404, G424, K427, S429, S591, and D593 for TycB_M3 were predicted to be key residues of the substrate-binding pocket. The binding energy of the G301A mutant (Fig. S14) with Phe (−11.9 kcal/mol) was significantly higher than that with Leu (−10.4 kcal/mol), indicating that it had a greater affinity for Phe. This suggests that G301A may preferentially recognize and activate Phe.

4. Discussion

Natural products have long been a valuable source of compounds used in medicine, agriculture, and biotechnology. The number of newly identified natural products increased markedly from the 1790s to the mid-1990s and has grown steadily since then [87]. In recent years, the discovery of novel AMPs has attracted increasing attention due to the global increase of antibiotic resistance and the urgent need for environmentally friendly alternatives in agriculture [88]. Genome mining has emerged as a powerful tool for uncovering novel natural products [89]. In this study, a genome mining-guided approach was used to explore the antimicrobial potential of B. brevis B011, a biocontrol strain with broad-spectrum antifungal activity. Using this strategy, we successfully identified several NRPS-associated BGCs and functionally and structurally elucidated both previously known and newly discovered AMPs.

This study indicates that edeine is the primary contributor to the antifungal activity of B. brevis B011, as deletion of the edeine BGC led to a significant reduction in antifungal activity. Edeine has broad-spectrum antifungal activity and operates via a dual mechanism of action, whereby it inhibits DNA polymerases at low concentrations and targets ribosomal function at higher concentrations [[90], [91], [92]]. This dual action enhances its antimicrobial potency and reduces the risk of resistance development. Edeine has been reported to eliminate plasmid-encoded resistance genes and inhibit bacterial cell division [40,41], highlighting its potential as a promising candidate for agricultural and medicinal applications.

B. brevis B011 produced tyrocidine related cyclic decapeptides detectable in both fermentation extracts and a one-pot in vitro system. Because the extract was complex and tyrocidines F–I were low-abundance with crowded peaks, we used defined substrates and purified TycA/B/C to obtain cleaner MS/MS data and strengthen the enzyme–product linkage. The establishment of this in vitro system provides an experimental basis for future A domain specificity engineering and combinatorial biosynthesis efforts aimed at altering amino-acid composition and potentially enhancing antimicrobial activity. Accurate mass analysis and MS/MS fragmentation patterns showed that the detected peptides correspond to tyrocidines F–I. Comparative analysis with previously reported data demonstrated that tyrocidine F represents a previously unreported analogue, whereas tyrocidines G, H and I were identical to streptocidins D, C and A, respectively, as described by Höltzel et al. (2001) [34]. The observed mass accuracy (<5 ppm) and consistent diagnostic fragment ions support the proposed primary structures. However, these structural assignments should be regarded as putative and are based on mass spectrometric evidence. In accordance with established nomenclature for cyclodecapeptide antibiotics, in which the amino acid residue at position 7 defines the peptide family (Tyr⁷ for tyrocidines, Trp⁷ for tryptocidines, and Phe⁷ for phenycidines), the detected peptides were named following this convention [84]. Accordingly, tyrocidine F retains its designation, tyrocidine G was renamed tryptocidine F, tyrocidine H was renamed tyrocidine G, and tyrocidine I was renamed tryptocidine G.

The successful in vitro reconstitution of the tyrocidine biosynthesis pathway validates the structural identities of the newly discovered derivatives and highlights the utility of one-pot, multi-enzyme synthetic systems as a powerful platform for natural product research. In contrast to conventional culture-dependent methods, this in vitro approach enables direct access to cryptic, low-abundance, and tightly regulated metabolites and simultaneously provides mechanistic insights into the modular architecture and functional logic of NRPS assembly lines [[93], [94], [95]]. Furthermore, this system can be leveraged to generate novel tyrocidine derivatives with structural diversity by introducing targeted mutations [61].

Amino acid substitution in tyrocidine derivatives can significantly affect their physicochemical properties, membrane-targeting abilities, and antimicrobial spectrum [61]. For instance, changes at positions involved in amphiphilicity or hydrogen bonding (such as Trp, Leu, or Tyr) modulate membrane insertion and the pore formation capacity, influencing their antifungal or antibacterial efficacy [30,73,74]. Thus, sequence modifications may influence their bioactivity spectrum, stability, and toxicity. Previous studies have shown that changes in key residues can alter the selectivity and potency of tyrocidine analogues [29,30].

NRPSs are large, multi-modular enzyme complexes that catalyze bioactive peptide biosynthesis in a template-directed fashion [96]. Among their modules, the A domain is responsible for the recognition and activation of specific amino acid substrates and is considered the “gatekeeper” of the NRPS assembly line [70]. The substrate specificity of the A domain is determined by a set of conserved residues known as the “specificity-conferring code.” Structural, sequence, and biochemical analyses of the Phe-activating A domain GrsA from gramicidin S synthetase revealed 10 active-site residues constituting the substrate specificity-conferring code (Fig. 6A,B) [85,86]. For example, the combination of T278 and A301 typically specifically recognizes and activates Phe (F), whereas substitutions, such as Met (M), Phe (F), Tyr (Y), or Leu (L), at position 278—particularly when position 301 is Gly (G)—favor Leu (L) selection [85,86]. Notably, site-directed mutagenesis at just one or two key positions significantly alters substrate preference and modifies the structures of the resulting peptides [[97], [98], [99]]. In non-ribosomal peptide luminmide synthesis, site mutagenesis of the A domain in module 3 (M3) of NRPS Plu3263 successfully reprogrammed substrate selectivity. The WT A3 domain predominantly recognized Phe (68%) and, to a lesser extent, Leu (29%), but the A301G mutant increased Leu specificity to 94.5%. The double mutant (C278 M/A301G) enhanced Leu recognition to 99.2%, effectively eliminating Phe incorporation [100]. In this study, multiple sequence alignment also revealed that Gly (G) was conserved at position 301 in TycB_M3 and TycC_M10 of B. brevis B011, suggesting its likely recognition and activation of Leu (Fig. S10). Furthermore, residues D240, G322, and S328 in TycB_M4 (Fig. S11) as well as A236, L239, A259, G301, E322, S328, and S329 in TycC_M7 (Fig. S12) may confer substrate selectivity for Phe or Trp and Tyr or Trp, respectively. Molecular docking revealed the Leu substrate binding pockets and specificity-conferring codes of the A domains in 011_TycB_M3. The Leu substrate specificity-conferring codes S401, Q404, G424, K427, S429, S591, and D593 for TycB_M3 were predicted to be key residues of the substrate-binding pocket. After mutating Gly424 to Ala (corresponding to G301A in multiple sequence alignment), the binding pocket volume decreased, and the introduction of a methyl side chain (from Ala, compared with a single hydrogen in Gly) increased steric hindrance [100]. This structural change hindered the accommodation of the isobutyl side chain of Leu but favored the binding of the less bulky phenyl ring of Phe. Docking binding energy calculations demonstrated the higher binding affinity of the G301A mutant for Phe than for Leu, indicating the enhanced substrate preference for Phe conferred by the G301A mutation. These findings provide a mechanistic explanation for the altered substrate preference conferred by the G301A mutation and highlight the structural plasticity of A domains in accommodating different amino acid side chains. Future work will involve high-throughput mutagenesis and activity assays to confirm these predictions and elucidate the substrate recognition logic of A domains. These findings lay the groundwork for the rational engineering of NRPS modules and the design of novel non-natural AMPs.

Combining genome mining, targeted gene disruption, metabolite profiling, and in vitro reconstitution supports functional assignment of selected BGCs to bioactive metabolites in B. brevis B011. Compared with conventional bioassay-guided isolation, which can be affected by scaffold rediscovery, low recovery of genuinely new compounds, and false positives [47], genome-informed analyses can prioritize testable candidates and guide validation when coupled with genetic and chemical evidence [48,[49], [50], [51]]. Under the tested conditions, the integration of bioinformatics prioritization, targeted mutagenesis, HPLC-MS/MS, and enzyme-based reconstitution established a genetic link between NRPS clusters and their AMPs in the biocontrol strain B. brevis B011.

Although AntiSMASH predicted a complete gramicidin BGC in the genome, the gramicidin-deficient mutant (011Δgra) showed no reduction in the antifungal activity compared with WT, and targeted LC–MS analysis failed to detect gramicidin-associated signals/target peaks under the tested conditions. These results indicate that gramicidin production is low or undetectable under the tested conditions, and thus likely contributes minimally to the observed antifungal activity. This could reflect condition-limited expression of the gramicidin BGC or constraints in precursor supply. These findings underscore the fundamental limitation of genome mining—the presence of a BGC does not guarantee metabolite production in the absence of experimental validation [47,48]. Building upon these findings, future work should focus on activating and characterizing additional secondary metabolites from B. brevis B011, especially those with potential bioactivity and unknown chemical scaffolds. Gene clusters with low expression or that have been silenced may be activated using cultivation-based strategies, such as nutrient supplementation, environmental adjustments (e.g., temperature, pH, and chemical signals), and co-cultivation with other microbes [101]. Alternatively, promoter replacement using constitutive or inducible elements can be applied to activate silenced BGCs [102]. These approaches will help explore the strain's metabolic potential and expand its application in natural product discovery.

Despite these advances, several limitations remain to be addressed. First, this study primarily focused on NRPS-type BGCs, leaving other predicted clusters, such as those encoding RiPPs, PKSs, and terpene-type metabolites, unexplored. These uncharacterized BGCs may harbor additional bioactive compounds with distinct chemical scaffolds and biological functions [[79], [80], [81], [82]]. Second, although the antifungal activities of the identified compounds were demonstrated under in vitro conditions, their efficacy under more complex ecological settings, such as greenhouse or field environments, remains to be validated. In particular, interactions among the host plant, pathogens, microbial community, and environmental factors can influence the biocontrol performance and stability of these metabolites [103]. Third, the transcriptional regulation underlying AMP biosynthesis in B. brevis remains poorly characterized, and further investigation into the control of silenced or conditionally expressed BGCs is warranted, particularly to enable the rational activation of silenced BGCs and fully harness the strain's biosynthetic potential [104]. Finally, it should be noted that the methanol-based extraction protocol used in this study may have imposed selectivity on the peptides recovered. Certain hydrophobic or aggregation-prone tyrocidine analogues may have been underrepresented or missed, and therefore the peptide profile reported here may not reflect the full diversity of cyclodecapeptides produced by the strain. Alternative extraction strategies could potentially reveal additional analogues [105,106].

This study presents B. brevis B011 as a potent biocontrol bacterium that can synthesize structurally diverse and biologically active AMPs. Genome mining and gene knockout identified edeine as the primary antifungal agent and tyrocidine as a secondary effector. One novel tyrocidine derivative (tyrocidine F), featuring a Leu residue at the third position, was putatively and preliminarily structurally characterized by HPLC–MS/MS, and its biosynthetic pathways was further supported by in vitro one-pot biosynthesis experiments. The tyrocidine F offer valuable scaffolds for developing next-generation antimicrobial agents with improved properties. Tryptocidine F, tyrocidine G and tryptocidine G were putatively identified as identical to streptocidins D, C and A, respectively; however, to the best of our knowledge, this study represents the first report of streptocidin-type members of the tyrocidine family produced by a Brevibacillus brevis strain, together with a characterization of their antimicrobial activity and the associated synthetase complex. In addition, molecular docking predicted a key specificity-conferring code (S401, Q404, G424, K427, S429, S591, and D593) in the A domain of TycB_M3, which conferred Leu specificity. This study has provided both theoretical and experimental evidence supporting the biocontrol potential of strain B011 in plant disease and demonstrated the utility of synthetic biology tools in expanding natural product diversity and accelerating the discovery of bioactive molecules from microbial resources. Future work should focus on rational design, structure–activity relationships, regulatory mechanisms governing BGC expression, and large-scale production strategies for practical field applications.

CRediT authorship contribution statement

Liang Zhang: Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Caichen Duan: Writing – original draft, Formal analysis, Conceptualization. Jing Fu: Visualization, Validation, Data curation. Cuiyang Zhang: Validation, Methodology. Ruoyi Lan: Methodology, Data curation. Hailin Cai: Supervision, Conceptualization. Qingshan Long: Visualization, Resources. Ying Tang: Visualization. Zhaohui Guo: Supervision. Jie Du: Validation, Resources, Funding acquisition. Wu Chen: Writing – review & editing, Writing – original draft, Resources, Funding acquisition, Data curation, Conceptualization. Qingshu Liu: Writing – review & editing, Writing – original draft, Resources, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Hailin Cai is currently employed by Changsha Branch of Hunan Tobacco Company.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Data availability

Data will be made available on request.